1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
2 Chinese Academy of Agricultural Sciences, Beijing 100081, China
Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice. About 30 bacterial blight resistance (R) genes (21 dominant R genes and 9 recessive ones) in rice have been identified. These R genes are known to act in a gene-for-gene manner and are the main sources for genetic improvement of rice for resistance to Xoo. Three cloned dominant R genes for resistance to Xoo belong to either LRR receptor kinase-like proteins or the NBS-LRR type, but little is known about the recessive R genes. To clone and characterize the recessive R genes at the molecular level, we fine-mapped a recessive gene, xa13, for bacterial blight resistance to a DNA fragment of 14.8 kb using map-based cloning strategy. Sequence analysis of this fragment indicated that this region contained only one complete open reading frame. These results will greatly facilitate the isolation and characterization of xa13.
Fine mapping of xa13 gene for bacterial blight resistance will facilitate the isolation and characterization of this recessive gene.
Bacterial blight, recessive resistance
Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice. More than 20 BB resistance (R) genes, included 9 recessive R genes, have been identified from cultivated rice and its wild relatives, or induced by mutagenesis (Lin et al. 1996; Nagato and Yoshimura 1998; Zhang et al. 1998; Khush and Angeles 1999; Chen et al. 2002; Lee et al. 2003; Yang et al. 2003). The dominant R genes included Xa1 to Xa28. Six of the recessive R genes, xa5, xa8, xa13, xa24, xa26 and xa28, occur naturally and confer race-specific resistance; the other 3, xa15, xa19 and xa20, are created by mutagenesis and each confers a wide spectrum of resistance to Xoo (Ogawa 1996; Lee et al. 2003). To date, only three dominant R genes for BB resistance, Xa1, Xa21 and Xa26, have been cloned. Xa21 and Xa26 encode leucine-rich repeat (LRR) receptor kinase-like proteins, while Xa1 encodes a nucleotide-binding site (NBS)-LRR protein (Song et al. 1995; Yoshimura et al. 1998; Sun et al. 2004).
The products of dominant R genes are presumed to function as receptors, which interact directly or indirectly with pathogen elicitors to initiate hypersensitive responses (Dangl and Jones. 2001). However, recessive R genes appear to function differently from most known dominant R genes that encode conserved domains. The products of 3 characterized recessive R genes, mlo, edr1 and RRS1-R, are all different. The dominant Mlo encode a transmembrane protein and EDR1 encodes a MAPKK kinase; both of the dominant proteins act as negative regulators of the defense response (Frye et al. 2001; Kim et al. 2002). Thus mlo and edr1 are the loss-function mutants. Another recessive R gene RRS1-R encodes a novel NBS-LRR-WRKY protein, which is presumed to function in the nucleus (Lahaye. 2002).
The recessive R gene xa13 specifically confers resistance to the Philippine Xoo race 6 that is compatible with most of the BB resistance genes. The xa13 was first characterized in the rice variety BJ1 and mapped on the long arm of rice chromosome 8 (Ogawa et al.1987; Zhang et al. 1996; Sanchez et al.1999). Unlike other recessive gene for BB resistance, such as xa5 showing partial dominance to the avirulent Xoo races, xa13 is a completely recessive gene and can strongly interact with xa5, Xa4 and Xa21 (Li et al. 2001). Small-scale gene expression studies and SSH cDNA library analysis revealed that some defense-responsive genes activated in xa13-mediated resistance is not involved in dominant R genes (Xa4, Xa10 and Xa26) mediated resistance (Zhou et al. 2002; Wen et al. 2003; Chu et al. 2004). These results indicate that xa13 may function differently from other R genes in initiating resistance against Xoo. Thus, molecular cloning of xa13 gene would greatly enhance our understanding of the defensive system in rice. We report here the fine mapping of xa13 to a 14.8-kb DNA fragment through map-based cloning strategy.
Two F2 populations, consisting of 6000 and 1972 individuals derived from a cross between susceptible rice line IR24 and its near-isogenic line IRBB13 carrying xa13, were evaluated for resistance to BB in 2000 and 2002, respectively. At booting stage, five of the uppermost fully expanded leaves of each plant were inoculated with Philippine race 6 (PXO99) of Xoo by the leaf-clipping method (Kauffman et al. 1973). Reaction to the pathogen was evaluated 21 days after inoculation.
A BAC library constructed with genomic DNA from rice cultivar Minghui 63 (indica) with an average insert length of 150 kb and a coverage equivalent to nine rice genomes (Peng et al. 1998) was used for the construction of the physical map.
A shotgun approach was used to determine the nucleotide sequence of BAC clones. The M13 universal forward and reverse primers and the BigDye Terminator Cycle Sequencing v2.0 kit (Applied Biosystems, Forster City, CA, USA) were used for sequencing. Sequences were assembled using the computer program Sequencher 4.1.2 (Gene Codes Coporation, Ann Arbor, MI, USA).
DNA sequence similarity analysis was performed using Blastn and Blastx programs (Altschul et al.1997). The gene was predicted by using Genscan program (Burge and Karlin, 1997) and Blast analysis against rice EST database (http://bioinformatics.hzau.edu.cn).
According to the genetic mapping results (Zhang et al. 1996; Sanchez et al. 1999), the xa13 gene was located on the long arm of rice chromosome 8 flanked by two RFLP markers, RG136 and R2027. Our results indicated that a PCR marker, E6a, located between RG136 and xa13, and a RFLP marker, S14003, flanked R2027. Thus, xa13 gene located between E6a and S14003. A physical map covering the region flanked by markers E6a and S14003 was constructed by chromosome walking using Minghui 63 BAC clones (Fig. 1). This map composed of 5 (22E06, 23P23, 30A21, 39N05 and 44E01) overlapping BAC clones. One clone, 44E01, showed sequence homology with two BAC clones from rice variety IR64, IR64a and IR64b locating in the xa13 region as reported by Sanchez et al. (1999).
Both Minghui 63 and IR64 are susceptible to Xoo strain PXO99. Thus they carry the allele of xa13 or the dominant gene Xa13. The two BAC clones, IR64a and IR64b from IR64, were sequenced. IR64a was about 90 kb in size and IR64b was 66.6 kb.
The F2 plants were examined using markers flanking xa13 to detect recombination events occurred between xa13 locus and markers. Twenty-six individuals from the 6000 F2 population grown in 2000 showed recombination between xa13 and marker E6a (Fig. 1). Six shotgun clones (RP3, RP4, RP5, RP7, RP8 and RP10) from IR64a and IR64b, two SSR markers (SR6 and SR11) and a PCR marker (ST9) were further used to screen the 26 recombinant individuals. Markers RP3, RP4, RP5, SR6 and RP7 detected 15 to 1 recombination events and the rest 4 markers co-segregated with xa13 (Fig. 1).
Figure 1. A contig map covering the xa13 region. The long horizontal line indicates the region containing xa13 locus. The short horizontal lines represent BAC clones. The numbers between molecular markers indicate the number of recombination events detected between xa13 locus and corresponding marker. The underlined numbers represent recombination events detected from the F2 population grown in 2002. Other numbers represent recombination events detected from he F2 population grown in 2000. The dashed vertical lines between markers and BAC clones indicate that hybridization between markers and BAC clones was verified.
In 2002, 1972 F2 plants were also examined with markers flanking xa13. Ten recombinant individuals were identified using marker E6a flanking one side of xa13 locus (Fig. 1). Recombination was further detected occurring between RP7 and xa13 in 2 of the 10 plants. But marker RP8 co-segregated with xa13 in the 10 plants detected by E6a. The marker SR11 on the other side of xa13 locus detected 8 recombinant individuals in the 1972 F2 plants. Two recombination events occurring between ST9 and xa13 loci were further identified in 2 of the 8 plants detected by SR11. Combining the results obtained from analysis of 36 and 8 F2 recombinants detected by E6a and SR11, respectively, the region containing the xa13 locus was narrowed down to a fragment flanked by RP7 and ST9. The sequences of BAC clones IR64a and IR64b revealed that this fragment was only 14.8 kb in length.
We have targeted a recessive gene, xa13, for bacterial blight resistance to a 14.8 kb DNA fragment. Sequence analysis of this fragment indicated that this region contained only one complete open reading frame. These results will greatly facilitate the isolation and characterization of this gene.
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